Bispecific antibodies and uses thereof

Abstract

The present invention provides a bispecific antibody and uses thereof, the bispecific antibody comprising an antigen-binding domain that specifically binds to EpCAM and an antigen-binding domain that specifically binds to CD3.

Description

[Technical field]

[0001] The present invention relates to the technical field of immunology, in particular to a bispecific antibody against EpCAM and CD3 and its use. [Background technology]

[0002] Bispecific antibodies (BsAbs), also called dual targeting antibodies, can simultaneously recognize and bind to two different antigens or epitopes and block two different signal pathways to fulfill their role. Compared with monoclonal antibodies (mAbs) that recognize a single antigen, bispecific antibodies have many advantages: (1) they can redirect specific immune effector cells to nearby tumor cells to enhance tumor killing, which cannot be achieved by combining mAb therapeutic strategies; (2) they increase binding specificity through the interaction of two different cell surface antigens; (3) they can reduce development costs, clinical trials, and regulatory review budgets compared to single antibody drug discovery in combination therapy; (4) they can simultaneously block two different pathways that exert unique or overlapping functions in pathogenic mechanisms compared to single antibody drugs in combination therapy.

[0003] Cancer and other diseases are both caused by multiple factors, and there are many signal pathways in the pathogenesis, and single-target immunotherapy cannot effectively kill target cells. Patients who receive mAb treatment may develop drug resistance or not respond to treatment. Therefore, bispecific antibodies have already become the main choice for the treatment of many diseases, such as cancer, inflammation, viral infection, and autoimmune diseases. However, bispecific antibodies do not exist in the natural environment and must be realized by recombinant DNA, cell fusion, or chemical binding techniques. Recombinant DNA technology is currently the most widely used technology for producing BsAb, but there are still many obstacles such as the difficulty of expressing BsAb, low yield, difficulty of purification, and poor stability, so it is very necessary to establish new bispecific antibodies, overcome the above obstacles, and establish corresponding immune killing animal models. The present invention provides a novel bispecific antibody and describes its pharmacological research method and results. Summary of the Invention [Problem to be solved by the invention]

[0004] The present invention develops a novel bispecific antibody that comprises an antigen-binding domain that specifically binds to EpCAM and an antigen-binding domain that specifically binds to CD3, and uses thereof. [Means for solving the problem]

[0005] Specifically, the present invention relates to the following aspects:

[0006] 1. A bispecific antibody, comprising an antigen-binding domain that specifically binds EpCAM and an antigen-binding domain that specifically binds CD3; wherein the antigen-binding domain that specifically binds to EpCAM is selected from the group consisting of: 1) An antigen-binding domain that specifically binds to EpCAM, comprising the following CDRs or variants thereof: (i) CDRH1, CDRH2 and CDRH3 contained in the heavy chain variable region shown in SEQ ID NO:14, and (ii) CDRL1, CDRL2 and CDRL3 contained in the light chain variable region shown in SEQ ID NO:13; Preferably, according to the Kabat sequence number system, the sequence of CDRL1 is shown in SEQ ID NO:32, the sequence of CDRL2 is shown in SEQ ID NO:33, the sequence of CDRL3 is shown in SEQ ID NO:34, the sequence of CDRH1 is shown in SEQ ID NO:35, the sequence of CDRH2 is shown in SEQ ID NO:36 and the sequence of CDRH3 is shown in SEQ ID NO:37; or 2) An antigen-binding domain that specifically binds to EpCAM, comprising the following CDRs or variants thereof: (i) CDRH1, CDRH2 and CDRH3 contained in the heavy chain variable region shown in SEQ ID NO:16, and (ii) CDRL1, CDRL2 and CDRL3 contained in the light chain variable region shown in SEQ ID NO:15; Preferably, according to the Kabat sequence number system and CDR definition system, the sequence of CDRL1 is shown in SEQ ID NO:38, the sequence of CDRL2 is shown in SEQ ID NO:39, the sequence of CDRL3 is shown in SEQ ID NO:40, the sequence of CDRH1 is shown in SEQ ID NO:41, the sequence of CDRH2 is shown in SEQ ID NO:42, and the sequence of CDRH3 is shown in SEQ ID NO:43; The antigen-binding domain that specifically binds to CD3 is selected from the group consisting of: 1) An antigen-binding domain that specifically binds to CD3, comprising the following CDRs or variants thereof: CDRH1, CDRH2 and CDRH3 comprised in the heavy chain variable region shown in SEQ ID NO:50, and CDRL1, CDRL2 and CDRL3 comprised in the light chain variable region shown in SEQ ID NO:51; Preferably, according to the Kabat sequence number system, the sequence of CDRH1 is shown in SEQ ID NO:44, the sequence of CDRH2 is shown in SEQ ID NO:45, and the sequence of CDRH3 is shown in SEQ ID NO:46, the sequence of CDRL1 is shown in SEQ ID NO:47, the sequence of CDRL2 is shown in SEQ ID NO:48, and the sequence of CDRL3 is shown in SEQ ID NO:49, or 2) An antigen-binding domain that specifically binds to CD3, comprising the following CDRs or variants thereof: CDRH1, CDRH2 and CDRH3 comprised in the heavy chain variable region shown in SEQ ID NO:58, and CDRL1, CDRL2 and CDRL3 comprised in the light chain variable region shown in SEQ ID NO:59; Preferably, the sequence of CDRH1 is shown in SEQ ID NO:52, the sequence of CDRH2 is shown in SEQ ID NO:53 and the sequence of CDRH3 is shown in SEQ ID NO:54, the sequence of CDRL1 is shown in SEQ ID NO:55, the sequence of CDRL2 is shown in SEQ ID NO:56 and the sequence of CDRL3 is shown in SEQ ID NO:57, according to the Kabat sequence number system, wherein the variants of the CDRs and the corresponding CDRs have 3, 2 or 1 amino acid difference, respectively, or have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, respectively, identity.

[0007] 2. The antigen-binding domain that specifically binds to EpCAM comprises the following heavy chain variable region and light chain variable region (or variants thereof): (i) a heavy chain variable region as set forth in SEQ ID NO:14, a light chain variable region as set forth in SEQ ID NO:13, or (ii) a heavy chain variable region as set forth in SEQ ID NO:16, a light chain variable region as set forth in SEQ ID NO:15, and wherein the antigen-binding domain that specifically binds to CD3 comprises the following heavy chain variable region and light chain variable region (or a variant thereof): (1) a heavy chain variable region set forth in SEQ ID NO:50 and a light chain variable region set forth in SEQ ID NO:51; or (2) a heavy chain variable region represented by SEQ ID NO: 58 and a light chain variable region represented by SEQ ID NO: 59; Preferably, the antigen-binding domain that specifically binds to EpCAM is in the form of a Fab fragment and the antigen-binding domain that specifically binds to CD3 is in the form of an ScFv, Preferably, wherein the antigen-binding domain that specifically binds to EpCAM comprises the following heavy chain variable region and light chain variable region (or variants thereof): (i) a heavy chain variable region as set forth in SEQ ID NO:14, a light chain variable region as set forth in SEQ ID NO:13, and wherein the antigen-binding domain that specifically binds to CD3 is selected from the group consisting of: (1) ScFv as shown in SEQ ID NO: 18 or a variant thereof; (2) ScFv as shown in SEQ ID NO: 19 or a variant thereof; wherein the variant and the corresponding variable region or ScFv have 3, 2 or 1 amino acid difference, respectively, or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, respectively.

[0008] 3. The bispecific antibody comprises: (1) A light chain-heavy chain pair that specifically binds to EpCAM, the light chain-heavy chain pair comprising or consisting of a light chain and a heavy chain, wherein the light chain comprises a light chain variable region and a light chain constant region (preferably, the sequence of which is set forth in any one of SEQ ID NOs: 1, 60 to 65), the heavy chain comprises a heavy chain variable region, a CH1 (preferably, the sequence of which is set forth in SEQ ID NO: 2) and a first Fc fragment, preferably the first Fc fragment comprises a hinge region (preferably, the sequence of which is set forth in SEQ ID NO: 3), a CH2 (preferably, the sequence of which is set forth in any one of SEQ ID NOs: 6, 7, 66 to 71) and a CH3a; (2) A fusion peptide that specifically binds to CD3, the fusion peptide comprising or consisting of an ScFv that specifically binds to CD3 and a second Fc fragment, preferably the ScFv comprising, in order from the N-terminus to the C-terminus, a heavy chain variable region, a connecting peptide (preferably, the sequence of which is shown in SEQ ID NO:4), and a light chain variable region, the second Fc fragment comprising, in order from the N-terminus to the C-terminus, a hinge region (preferably, the sequence of which is shown in SEQ ID NO:3), a CH2 (preferably, the sequence of which is shown in any one of SEQ ID NOs:6, 7, 66 to 71), and a CH3b, preferably the C-terminus of the light chain variable region and the hinge region of the second Fc fragment are linked by a connecting peptide (preferably, the sequence of which is shown in SEQ ID NO:5); Preferably, the first Fc fragment and the second Fc fragment are human or humanized Fc fragments, such as human IgG Fc fragments, such as IgG1, IgG2, IgG3, IgG4, IgG5 Fc fragments; Preferably, compared to a wild-type antibody, the first Fc fragment and / or the second Fc fragment comprises one or more substitutions that form a mortar-and-pestle structure pair between the heavy chain and the fusion peptide, for example, T366 on one CH3 domain is replaced with a relatively large amino acid residue such as tyrosine (Y) or tryptophan (W), and Y407 on the other CH3 domain is replaced with a relatively small amino acid residue such as threonine (T), alanine (A) or valine (V), for example comprising one or more substitutions in Table 6; Preferably, the first Fc fragment and / or the second Fc fragment comprises one or more substitutions, 1) the substitutions form a salt bridge pair between the heavy chain and the fusion peptide, for example, one CH3 domain comprises one or more substitutions and is substituted by an amino acid residue that is positively charged under physiological conditions, and the other CH3 domain comprises one or more substitutions and is substituted by an amino acid residue that is negatively charged under physiological conditions, for example, the positively charged amino acid residue is arginine (R), histidine (H) or lysine (K), for example, the negatively charged amino acid residue ... negatively charged amino acid residue is substituted by an amino acid residue that is negatively charged under physiological conditions, for example, the positively charged amino acid residue is arginine (R), histidine (H) or lysine (K), for example, the negatively charged amino acid residue is substituted by an amino acid residue that is negatively charged under physiological conditions, for example, the negatively charged amino acid residue is substituted by an amino acid residue that is negatively charged under physiological conditions, for example, the negatively charged amino acid residue is substituted by an amino acid residue that is negatively charged under physiological conditions, for example, the negatively charged amino acid residue is substituted by an amino acid residue that is negatively charged under physiological conditions, for example, the 1) the amino acid residue is asparagine (D) or glutamic acid (E), e.g., the substituted amino acid residues include one or more of D356, L368, K392, D399 and K409, e.g., one or more substitutions in Table 7; 2) the substitution forms a disulfide bond between the heavy chain and the fusion peptide, e.g., a substitution in Table 8; and / or 3) the substitution significantly reduces the binding ability between Fc and Protein A, e.g., H435 and Y436 on one CH3 domain are substituted with arginine and phenylalanine, respectively, as shown in Table 9; Preferably, where: a) the CH3b of the fusion peptide and the CH3a of the heavy chain have a substitution pair that forms a pestle-and-mortar structure; b) the CH3b of the fusion peptide and the CH3a of the heavy chain have a substitution pair that forms an ionic bond; c) the CH3b of the fusion peptide and the CH3a of the heavy chain have a substitution pair that forms a disulfide bond; and / or d) the CH3b of the fusion peptide and the CH3a of the heavy chain have substitutions that result in a reduced ability to bind protein A; Preferably, CH1 comprises the sequence of SEQ ID No: 2 and / or CL comprises a sequence selected from any one of SEQ ID Nos: 1, 60 to 65, Preferably, the first Fc fragment and / or the second Fc fragment comprises a CH2 of a sequence selected from any one of SEQ ID Nos: 6, 7, 66 to 71 and / or a CH3 of a sequence selected from any one of SEQ ID Nos: 8, 9, 11, 12, 72 to 76, Preferably, the sequences of CH3a and CH3b are selected from the group consisting of: (1) one sequence of which is shown in SEQ ID NO:8 and another sequence of which is shown in SEQ ID NO:11; (2) one sequence of which is shown in SEQ ID NO:9 and another sequence of which is shown in SEQ ID NO:12; (3) one sequence of which is shown in SEQ ID NO:72 and another sequence of which is shown in SEQ ID NO:74; (4) one sequence of which is shown in SEQ ID NO:9 and another sequence of which is shown in SEQ ID NO:75; (5) one sequence of which is shown in SEQ ID NO: 73 and another sequence of which is shown in SEQ ID NO: 76; Preferably, the bispecific antibody is selected from the group consisting of: (1) it comprises or consists of a fusion peptide, a heavy chain and a light chain, wherein the fusion peptide comprises or consists of SEQ ID NO:18, SEQ ID NO:5, SEQ ID NO:3, SEQ ID NO:6 and SEQ ID NO:11, the heavy chain comprises or consists of SEQ ID NO:14, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:6 and SEQ ID NO:8, and the light chain comprises or consists of SEQ ID NO:13 and SEQ ID NO:1; (2) it comprises or consists of a fusion peptide, a heavy chain and a light chain, wherein the fusion peptide comprises or consists of SEQ ID NO:19, SEQ ID NO:5, SEQ ID NO:3, SEQ ID NO:6 and SEQ ID NO:11, the heavy chain comprises or consists of SEQ ID NO:14, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:6 and SEQ ID NO:8, and the light chain comprises or consists of SEQ ID NO:13 and SEQ ID NO:1; (3) it comprises or consists of a fusion peptide, a heavy chain and a light chain, wherein the fusion peptide comprises or consists of SEQ ID NO:18, SEQ ID NO:5, SEQ ID NO:3, SEQ ID NO:6 and SEQ ID NO:11, the heavy chain comprises or consists of SEQ ID NO:16, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:6 and SEQ ID NO:8, and the light chain comprises or consists of SEQ ID NO:15 and SEQ ID NO:1; (4) it comprises or consists of a fusion peptide, a heavy chain and a light chain, wherein the fusion peptide comprises or consists of SEQ ID NO:19, SEQ ID NO:5, SEQ ID NO:3, SEQ ID NO:6 and SEQ ID NO:11, the heavy chain comprises or consists of SEQ ID NO:16, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:6 and SEQ ID NO:8, and the light chain comprises or consists of SEQ ID NO:15 and SEQ ID NO:1; (5) it comprises or consists of a fusion peptide, a heavy chain and a light chain, wherein the fusion peptide comprises or consists of SEQ ID NO:18, SEQ ID NO:5, SEQ ID NO:3, SEQ ID NO:7 and SEQ ID NO:12, the heavy chain comprises or consists of SEQ ID NO:14, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:7 and SEQ ID NO:9, and the light chain comprises or consists of SEQ ID NO:13 and SEQ ID NO:1; (6) it comprises or consists of a fusion peptide, a heavy chain and a light chain, wherein the fusion peptide comprises or consists of SEQ ID NO:19, SEQ ID NO:5, SEQ ID NO:3, SEQ ID NO:7 and SEQ ID NO:12, the heavy chain comprises or consists of SEQ ID NO:14, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:7 and SEQ ID NO:9, and the light chain comprises or consists of SEQ ID NO:13 and SEQ ID NO:1; (7) It comprises or consists of a fusion peptide, a heavy chain and a light chain, wherein the fusion peptide comprises or consists of SEQ ID NO:18, SEQ ID NO:5, SEQ ID NO:3, SEQ ID NO:6 and SEQ ID NO:8, the heavy chain comprises or consists of SEQ ID NO:14, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:6 and SEQ ID NO:11, and the light chain comprises or consists of SEQ ID NO:13 and SEQ ID NO:1; (8) It comprises or consists of a fusion peptide, a heavy chain and a light chain, wherein the fusion peptide comprises or consists of SEQ ID NO:19, SEQ ID NO:5, SEQ ID NO:3, SEQ ID NO:6 and SEQ ID NO:8, the heavy chain comprises or consists of SEQ ID NO:14, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:6 and SEQ ID NO:11, and the light chain comprises or consists of SEQ ID NO:13 and SEQ ID NO:1; (9) It comprises or consists of a fusion peptide, a heavy chain and a light chain, wherein the fusion peptide comprises or consists of SEQ ID NO:18, SEQ ID NO:5, SEQ ID NO:3, SEQ ID NO:6 and SEQ ID NO:8, the heavy chain comprises or consists of SEQ ID NO:16, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:6 and SEQ ID NO:11, and the light chain comprises or consists of SEQ ID NO:15 and SEQ ID NO:1; (10) It comprises or consists of a fusion peptide, a heavy chain and a light chain, wherein the fusion peptide comprises or consists of SEQ ID NO:19, SEQ ID NO:5, SEQ ID NO:3, SEQ ID NO:6 and SEQ ID NO:8, the heavy chain comprises or consists of SEQ ID NO:16, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:6 and SEQ ID NO:11, and the light chain comprises or consists of SEQ ID NO:15 and SEQ ID NO:1; (11) It comprises or consists of a fusion peptide, a heavy chain and a light chain, wherein the fusion peptide comprises or consists of SEQ ID NO:18, SEQ ID NO:5, SEQ ID NO:3, SEQ ID NO:7 and SEQ ID NO:9, the heavy chain comprises or consists of SEQ ID NO:14, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:7 and SEQ ID NO:12, and the light chain comprises or consists of SEQ ID NO:13 and SEQ ID NO:1; (12) The bispecific antibody according to item 1 or 2, which comprises or consists of a fusion peptide, a heavy chain and a light chain, wherein the fusion peptide comprises or consists of SEQ ID NO:19, SEQ ID NO:5, SEQ ID NO:3, SEQ ID NO:7 and SEQ ID NO:9, the heavy chain comprises or consists of SEQ ID NO:14, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:7 and SEQ ID NO:12, and the light chain comprises or consists of SEQ ID NO:13 and SEQ ID NO:1.

[0009] 4. A nucleic acid composition comprising a nucleic acid sequence encoding the bispecific antibody according to any one of items 1 to 3, preferably comprising: The nucleic acid composition comprises: a) a first expression vector comprising a first nucleic acid encoding an antigen-binding domain or a light chain-heavy chain pair that specifically binds to EpCAM as defined in any one of items 1 to 3; b) a second expression vector comprising a second nucleic acid encoding an antigen-binding domain or a fusion peptide that specifically binds to CD3 as defined in any one of items 1 to 3.

[0010] 5. An expression vector comprising the nucleic acid composition of item 4.

[0011] 6. A host cell comprising the expression vector of item 5.

[0012] 7. A method for treating cancer (EpCAM-positive tumors, such as colorectal cancer, gastric cancer, breast cancer, ovarian cancer, lung cancer (e.g., non-small cell lung cancer), prostate cancer, pancreatic cancer, liver cancer, retinoblastoma, esophageal cancer, renal cancer, renal clear cell tumor, skin squamous carcinoma, skin basal cell carcinoma, sarcoma, nasal glioma, craniopharyngeal duct tumor, thyroid cancer, cholangiocarcinoma, bladder cancer, head and neck tumor, cervical cancer, or oral cancer) and / or malignant ascites, malignant effusion, malignant pleural effusion, etc., wherein the dosage form of the pharmaceutical composition comprises a gastrointestinal dosage form or a parenteral dosage form, and more preferably the dosage form of the pharmaceutical composition is an injectable formulation, including intravenous injection, intravenous drip, subcutaneous injection, local injection, intramuscular injection, intratumoral injection, intraperitoneal injection, intracranial injection, or intracavity injection.

[0013] 8. A conjugate or fusion protein comprising the bispecific antibody according to any one of items 1 to 3, preferably comprising a substance A conjugated or fused to said bispecific antibody, said substance A being selected from the group consisting of a therapeutic agent, a drug precursor, a protein (e.g. an enzyme), a virus, a lipid, a biological response modifier (e.g. an immunomodulator), PEG, a hormonal agent, an oligonucleotide, a diagnostic agent, a cytotoxic agent, which may be a drug or a toxin, an ultrasound enhancing agent, a non-radioactive marker, capable of detecting a marker such as a chemiluminescent marker compound (e.g. luminol, isoluminol, a thermoactive acridinium ester, an imidazole, an acridinium salt, an oxalate ester), or a fluorescent metal (e.g. 152Eu, or a lanthanide marker).

[0014] 9. A kit comprising the bispecific antibody according to any one of items 1 to 3, and optionally a drug (e.g., a small molecule drug or a polymer drug) for treating cancer (EpCAM-positive tumor, for example, colon cancer, gastric cancer, breast cancer, ovarian cancer, lung cancer (e.g., non-small cell lung cancer), prostate cancer, pancreatic cancer, liver cancer, retinoblastoma, esophageal cancer, renal cancer, renal clear cell tumor, skin squamous cancer, skin basal cell carcinoma, sarcoma, nasal glioma, craniopharyngeal duct tumor, thyroid cancer, cholangiocarcinoma, bladder cancer, head and neck tumor, cervical cancer, or oral cancer, etc.) and / or malignant ascites, malignant retention fluid, malignant pleural effusion, etc.

[0015] 10. Use of the bispecific antibody according to any one of items 1 to 3 in the treatment of cancer, or in the manufacture of a drug or kit for the treatment of cancer and / or malignant ascites, malignant pleural effusion, or the like, wherein the cancer, e.g., EpCAM-positive tumor, is, for example, colorectal cancer, gastric cancer, breast cancer, ovarian cancer, lung cancer (e.g., non-small cell lung cancer), prostate cancer, pancreatic cancer, liver cancer, retinoblastoma, esophageal cancer, renal cancer, renal clear cell tumor, skin squamous cancer, skin basal cell carcinoma, sarcoma, nasal glioma, craniopharyngeal duct tumor, thyroid cancer, cholangiocarcinoma, bladder cancer, head and neck tumor, cervical cancer, or oral cancer.

[0016] 11. A method for treating cancer and / or malignant ascites, malignant retention fluid, or malignant pleural effusion, comprising administering to a subject a therapeutically effective amount of the bispecific antibody according to any one of items 1 to 3, wherein the EpCAM-positive tumor is, for example, colorectal cancer, gastric cancer, breast cancer, ovarian cancer, lung cancer (e.g., non-small cell lung cancer), prostate cancer, pancreatic cancer, liver cancer, retinoblastoma, esophageal cancer, renal cancer, renal clear cell tumor, skin squamous carcinoma, skin basal cell carcinoma, sarcoma, nasal glioma, craniopharyngeal duct tumor, thyroid cancer, cholangiocarcinoma, bladder cancer, head and neck tumor, cervical cancer, or oral cancer.

[0017] It should be understood that within the scope of the present invention, the above technical features of the present invention and the technical features specifically described below (e.g., in the Examples) can be combined with each other to form new or preferred technical solutions, which will not be described here due to space limitations.

[0018] The terms of the present invention have the ordinary meanings as understood by those skilled in the art. The definitions of terms used herein are used to include all meanings when a term has more than one definition, as used and / or permitted in the art.

[0019] Those skilled in the art can understand that the CDR region of an antibody is responsible for the binding specificity of the antibody to the antigen. When the antibody heavy and light chain variable region sequences are known, there are currently several methods for determining the antibody CDR region, including Kabat, IMGT, Chothia and AbM numbering systems. However, each application of the definition of CDRs of various antibodies or variants thereof is within the scope of the term as defined and used herein. When the variable region amino acid sequence of the antibody is given, those skilled in the art can usually determine a particular CDR without relying on any experimental data other than the sequence itself.

[0020] As used herein, "antibody" or "antigen-binding fragment" refers to a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen. The term "antibody" is used broadly and includes immunoglobulins or antibody molecules, including monoclonal or polyclonal human, humanized, conjugated and chimeric antibodies, as well as antibody fragments. Thus, the term "antibody" includes any protein or peptide that includes a specific molecule, which includes at least a portion of an immunoglobulin molecule that has the biological activity of binding to an antigen. Examples of such situations include, but are not limited to, the complementarity determining regions (CDRs) of a heavy or light chain or a ligand-binding portion thereof, a heavy or light chain variable region, a heavy or light chain constant region, a framework (FR) region or any portion thereof, or at least a portion of a binding protein. In the present invention, an antibody includes murine, chimeric, humanized or fully human antibodies made using techniques familiar to those skilled in the art. Recombinant antibodies, such as chimeric and humanized monoclonal antibodies, including human and non-human portions, can be made using DNA recombinant techniques well known in the art. The immunoglobulin or antibody molecules of the present application may be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule.

[0021] The term "antibody fragment" or "antigen-binding fragment" includes, but is not limited to, F(ab')2, F(ab)2, Fab', Fab, Fv, Fd, dAb, Fab / c, complementarity determining region (CDR) fragments, single chain Fvs (ScFv), disulfide-stabilized Fv fragment (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), double-chain antibodies (Diabodies), disulfide-stabilized double-chain antibodies (ds-Diabodies), ScFv multimers (such as ScFv dimers, ScFv trimers), multispecific antibodies formed from a portion of an antibody comprising one or more CDRs, nanobodies, single domain antibodies (sdab), domain antibodies, bivalent domain antibodies, or any other antibody fragment that binds to an antigen but does not comprise the complete antibody structure. Regardless of structure, an antigen-binding fragment includes a polypeptide or polypeptide complex capable of binding to the same antigen that binds to the parent antibody or parent antibody fragment. The term "antibody fragment" includes aptamers, aptamer spiegelmers, and diabodies. The term "antibody fragment" also includes synthetic or genetically engineered proteins that, like antibodies, can bind to a specific antigen to form a complex. Generally, an antibody fragment has at least about 50 consecutive amino acids of an antibody of the invention, preferably at least about 50 consecutive amino acids, more preferably at least about 80 consecutive amino acids, and optimally at least about 100 consecutive amino acids.

[0022] "Single chain variable fragment" or "ScFv" refers to a fusion protein of the variable regions of the heavy (VH) and light (VL) chains of an immunoglobulin. In some embodiments, these regions are linked by a short linker peptide of 10 to about 25 amino acids. The linker can be glycine-rich for flexibility or can contain serine or threonine for solubility, and can link the N-terminus of VH to the C-terminus of VL, or vice versa. The protein retains the properties of an original immunoglobulin by simply removing the constant regions and introducing the linker. ScFv molecules are known in the art, such as those described in U.S. Pat. No. 5,892,019.

[0023] The antigen-binding domain that binds EpCAM and CD3 is a Fab, or ScFv, or a non-covalent bond (Fv) between the heavy chain variable region (VH) and the light chain variable region (VL). Any of the above antibodies or polypeptides may further comprise additional polypeptides, such as a signal peptide at the antibody N-terminus to induce secretion, or other heterologous polypeptides as described herein, such as a 6xHis tag for purification. The present invention includes complete antibodies, as well as immunologically active antibody fragments or fusion proteins formed with antibodies and other sequences. The present invention further provides other proteins or fusion expression products comprising the antibodies of the present invention. Specifically, the present invention includes any protein or protein conjugate and fusion expression product (i.e., immunoconjugates and fusion expression products) having heavy and light chains containing variable regions, so long as the variable regions are identical or have at least 90% homology, preferably at least 95% homology, and optimally 96%, 97%, 98% or 99% or more homology to the variable regions of the heavy and light chains of the antibodies of the present invention. Thus, the present invention includes molecules having the light and heavy chain variable regions of a monoclonal antibody having CDRs, so long as the CDRs have 90% or more (preferably 95% or more, and optimally 96%, 97%, 98%, or 99% or more) homology with the CDRs of the present invention.

[0024] The present invention further includes fragments, variants, derivatives and analogs of said antibodies. The antibodies, antigen-binding fragments, variants or derivatives thereof of the present application include, but are not limited to, polyclonal antibodies, monoclonal antibodies, multispecific antibodies (e.g., bispecific antibodies, trispecific antibodies, etc.), human antibodies, animal-derived antibodies, humanized antibodies, primatized antibodies or chimeric antibodies, CDR-grafted and / or modified antibodies, single chain antibodies (e.g., ScFv), double chain antibodies, antigen epitope-binding fragments such as Fab, Fab' and F(ab')2, Fd, Fv, single chain Fv (ScFv), single chain antibodies, disulfide-linked Fv (dsFv), fragments comprising VL or VH domains, fragments produced from a Fab expression library, and anti-idiotypic (anti-Id) antibodies. The antibody fragment, antigen-binding fragment, derivative or analog of the present invention may be (i) a polypeptide in which one or more conservative or non-conserved amino acid residues (preferably conservative amino acid residues) have been substituted (wherein such substituted amino acid residues may or may not be codon-encoded), or (ii) a polypeptide having a substitution in one or more amino acid residues, or (iii) a polypeptide formed by fusing the mature polypeptide with another compound (a compound that extends the half-life of the polypeptide, such as polyethylene glycol), or (iv) a polypeptide formed by fusing an additional amino acid sequence to such a polypeptide sequence (e.g., a preamble sequence or a secretory sequence, or a sequence for purifying such a polypeptide or a proprotein sequence, or a fusion protein with a 6xHis tag). Based on the teachings of the present specification, these fragments, derivatives and analogs are within the scope well known to those skilled in the art.

[0025] The antibody of the present invention refers to a polypeptide having the binding activity between human EpCAM and CD3 and comprising the above CDR region. The term further includes mutant forms of the polypeptide having the same function as the antibody of the present invention and comprising the above CDR region. These mutant forms include, but are not limited to, deletion, insertion and / or substitution of one or more amino acids (generally 1 to 50, preferably 1 to 30, more preferably 1 to 20, optimally 1 to 10) and addition of one or more amino acids (generally 20 or less, more preferably 10 or less, more preferably 5 or less) at the C-terminus and / or N-terminus. For example, in this field, substitution with an amino acid having close or similar performance usually does not change the function of the protein. Also, for example, addition of one or several amino acids to the C-terminus and / or N-terminus usually does not change the function of the protein. The term further includes active fragments and active derivatives of the antibody of the present invention. Variant forms of the polypeptide include homologous sequences, conservative variants, coordinate variants, naturally occurring variants, induced variants, proteins encoded by DNA capable of hybridizing to the coding DNA of the antibody of the present invention under high or low stringency conditions, and polypeptides or proteins obtained using antisera against the antibody of the present invention.

[0026] The antibody of the present invention may be (i) a polypeptide in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, or (ii) a polypeptide having a substitution in one or more amino acid residues, or (iii) a polypeptide formed by fusing the mature polypeptide with another compound (a compound that extends the half-life of the polypeptide, such as polyethylene glycol), or (iv) a polypeptide formed by fusing an additional amino acid sequence to such a polypeptide sequence (e.g., a preamble sequence or a secretory sequence, or a sequence for purifying such a polypeptide or a proprotein sequence, or a fusion protein with a 6xHis tag). Based on the teachings of the present specification, these fragments, derivatives and analogs are within the scope well known to those skilled in the art.

[0027] "Conservative amino acid substitution" means that the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art and include basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, non-essential amino acid residues in immunoglobulin polypeptides are preferably replaced with other amino acid residues from the same side chain family. In some alternative embodiments, the amino acid string may be replaced with a structurally similar amino acid string, the latter differing in order and / or side chain family composition.

[0028] Non-limiting examples of conservative amino acid substitutions are shown in the following table, where a similarity score of 0 or greater indicates a conservative substitution between the two amino acids. [Table 1] TIFF2024540461000002.tif31169

[0029] In some embodiments, the conservative substitutions are preferably those in which one amino acid residue within the following groups (a)-(e) is substituted with another amino acid residue within the same group: (a) small aliphatic, non-polar or weakly polar residues: Ala, Ser, Thr, Pro and Gly; (b) polar, negatively charged residues and their (uncharged) amides: Asp, Asn, Glu and Gln; (c) polar, positively charged residues: His, Arg and Lys, (d) large aliphatic, non-polar residues: Met, Leu, Ile, Val and Cys, and (e) aromatic residues: Phe, Tyr and Trp.

[0030] Particularly preferred conservative substitutions are: Ala to Gly or Ser; Arg to Lys; Asn to Gln or His; Asp to Glu; Cys to Ser; Gln to Asn; Glu to Asp; Gly to Ala or Pro; His to Asn or Gln; Ile to Leu or Val; Leu or substitute Ile or Val; Lys for Arg; Gln for Glu; Met for Leu; Tyr for Ile; Phe for Met; Leu for Tyr; Ser for Thr; Thr for Ser; Trp for Tyr; Tyr for Trp; and / or Phe for Val; Ile for Leu or Leu.

[0031] The Fc amino acid numbering follows the Kabat numbering system described by Kabat et al. in the U.S. Department of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983). The specific numbers are as follows: [Table 2]

[0032] Where: The amino acids 221 to 227 are a hinge domain. Amino acids 228 to 340 are the second constant region CH2 domain of the heavy chain; Amino acids 341 to 447 represent the third constant region CH3 domain of the heavy chain.

[0033] Antibodies may be modified to improve heterodimer pairing efficiency. For example, in some embodiments, the Fc fragment of the monovalent heavy chain and / or the Fc fragment of the fusion peptide may contain one or more substitutions compared to the wild-type antibody fragment, which form a knob-into-holes structure pair between the substitutions. Knob-into-holes structures are known in the art. See, for example, Ridgway et al., "'Knob-into-holes' engineering of antibody CH3 domains for heavy chain heterodimerization," Protein Engineering 9(7):617-21 (1996).

[0034] In one embodiment, T366 on one CH3 domain is substituted with a relatively large amino acid residue such as tyrosine (Y) or tryptophan (W), and Y407 on another CH3 domain may be substituted with a relatively small amino acid residue such as threonine (T), alanine (A), or valine (V). [Table 3]

[0035] In one embodiment, one of the CH3 domains comprises one or more substitutions with amino acid residues that are positively charged under physiological conditions, and another CH3 domain comprises one or more substitutions with one or more amino acid residues that are negatively charged under physiological conditions. In one embodiment, the positively charged amino acid residue may be arginine (R), histidine (H) or lysine (K). In another embodiment, the negatively charged amino acid residue may be aspartic acid (D) or glutamic acid (E). Amino acid residues that may be substituted include, but are not limited to, D356, L368, K392, D399 and K409. [Table 4]

[0036] In one embodiment, S354 on one CH3 domain was substituted with a cysteine ​​and Y349 on the other CH3 domain was also substituted with a cysteine, such that the two substituted residues formed a disulfide bond. [Table 5]

[0037] In one embodiment, H435 and Y436 on one CH3 domain are substituted with arginine and phenylalanine, respectively, which significantly reduces the binding ability between Fc and Protein A, thereby providing different Protein A binding activities between the heterodimer and homodimer, allowing them to be easily separated during affinity chromatography. [Table 6]

[0038] In one preferred embodiment of the present invention, the CH3 amino acid sequences of the Fc forming the heterodimer are shown in the table below: [Table 7] TIFF2024540461000009.tif230169 TIFF2024540461000010.tif55169

[0039] One embodiment of the present application provides a heterodimeric antibody comprising two different antigen-binding polypeptide units, which in some embodiments differ in size from the corresponding homodimer, and the size difference can be exploited to facilitate separation of the heterodimer and homodimer.

[0040] In some embodiments, as in Figure 1, one of these two antigen-binding polypeptide units comprises a light-heavy chain pair similar to a wild-type antibody. Throughout the application, this unit is also referred to as the "monovalent unit". In some embodiments, as in Figure 1, the other antigen-binding polypeptide unit comprises a single-chain variable fragment (ScFv). Such a ScFv can be fused to the N-terminus of the constant fragment (Fc) of an antibody, called a fusion peptide. Throughout the application, this fusion peptide is also referred to as the "single-chain unit".

[0041] Any of the above antibodies or polypeptides may further comprise additional polypeptides, such as the encoded polypeptides described herein, a signal peptide of the antibody constant region to direct secretion, or other heterologous polypeptides described herein. The antibodies described herein may be modified such that their amino acid sequences differ from naturally occurring binding polypeptides. For example, a polypeptide or amino acid sequence derived from a particular protein may be similar to the starting sequence, e.g., may have a certain percentage of identity with the starting sequence, e.g., 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity with the starting sequence. Additionally, nucleotide or amino acid substitutions, deletions, or insertions may be made to make conservative substitutions or modifications in "non-essential" amino acid regions. For example, a polypeptide or amino acid sequence derived from a particular protein may be the same as the starting order, except for one or more independent amino acid substitutions, insertions, or deletions, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more independent amino acid substitutions, insertions, or deletions. In particular embodiments, a polypeptide or amino acid sequence derived from a particular protein has 1-5, 1-10, 1-15, or 1-20 independent amino acid substitutions, insertions, or deletions relative to the starting sequence.

[0042] The term "detectable tag" as used herein means a directly or indirectly detectable compound or composition that is directly or indirectly bound to a composition to be detected (e.g., a polynucleotide or a protein such as an antibody) to obtain a "tagged" composition. The term further provides a sequence that binds to the polynucleotide that provides a signal such as green fluorescent protein (GFP) upon expression of the inserted sequence. The tag itself (e.g., a radioisotope tag or a fluorescent tag) can be detected, or in the case of an enzymatic tag, can catalyze a chemical change in a substrate compound or composition, which modification can be detected. The tag can be used for small-scale detection or is more suitable for high-throughput screening. Similarly, suitable tags include, but are not limited to, radioisotopes, fluorescent dyes, chemiluminescent compounds, dyes, and proteins (including enzymes). The tag can be detectable only and can also be quantified. A detectable only reaction generally includes a reaction that only confirms its presence, whereas a quantifiable reaction generally includes a reaction that has a quantifiable (e.g., digitally reportable) value, such as intensity, polarization, and / or other property. In luminescent or fluorescent assays, the detectable reaction can directly employ a luminescent or fluorescent group that is actually bound to the analytical component, or indirectly employ a luminescent or fluorescent group that is bound to another (e.g., a reporter molecule or therapeutic) component.

[0043] In some embodiments, the antibodies of the invention can be conjugated to a therapeutic agent (e.g., chemotherapy drugs such as cisplatin, carboplatin, etc.), drug precursors, peptides, proteins, enzymes, viruses, lipids, biological response modifiers, drugs, or PEG. The antibodies of the invention can be conjugated or fused to a therapeutic agent, including detectable markers such as radioactive markers, immunomodulators, hormones, enzymes, oligonucleotides, photoactive therapeutic or diagnostic agents, cytotoxic agents, which may be drugs or toxins, ultrasound enhancing agents, non-radioactive markers, combinations thereof, and other such moieties known in the art.

[0044] In some examples, the antigen-binding polypeptide comprises an amino acid sequence or one or more groups not normally bound to antibodies. For example, the single chain Fv antibody fragment of the present application may comprise a flexible linker sequence or a functional group that may be modified to attach (e.g., polyethylene glycol (PEG), a drug, a toxin, or a marker). The antibodies, variants or derivatives thereof of the present application include modified derivatives, i.e., any type of molecule may be covalently attached to the antibody, where the covalent attachment does not inhibit the antibody from binding to the antigen epitope. Additionally, the antibody may comprise one or more non-classical amino acids.

[0045] It should be noted that implicit numerical limitations of an entity refer to one or more of that entity, e.g., a "multifunctional antibody" should be understood to refer to eleven or more multifunctional antibodies. Similarly, the terms "one or more" and "at least one" in implicit numerical limitations are used interchangeably herein.

[0046] The term "treatment" as used herein refers to therapeutic treatment and prophylactic or preventative measures, where an undesirable physiological change or disease, such as the progression of cancer, is prevented or delayed (alleviated) in a subject. Beneficial or desired clinical results include, but are not limited to, alleviating symptoms, whether detectable or not, reducing the extent of the disease, stabilizing (e.g., not worsening) the state of the disease, delaying or slowing the progression of the disease, improving or alleviating the state of the disease, and alleviating (partially or completely). "Treatment" may also mean prolonging survival compared to expected survival in the absence of treatment. Situations requiring treatment include those where a disease or condition is already present, or where a disease or condition is susceptible to developing, or where a disease or condition can be prevented.

[0047] The so-called "subject" or "individual" or "animal" or "patient" or "mammal" refers to any subject requiring diagnosis, prognosis, or treatment, particularly a mammalian subject. Mammalian subjects include humans, domestic animals, farm animals, zoos, exercise yards, or dogs, cats, guinea pigs, rabbits, rats, mice, rats, horses, cows, dairy cows, primates (e.g., humans, such as cynomolgus monkeys, macaques, baboons, monkeys, chimpanzees, etc.), and the like.

[0048] The antigen-binding polypeptides, variants or derivatives described herein can be used in several therapeutic and diagnostic methods related to cancer or infectious diseases. The present application also relates to antibody-based therapies, including administering the bispecific antibodies of the present application to patients, such as animals, mammals, and humans, to treat one or more diseases or conditions described herein. Therapeutic agents of the present application include, but are not limited to, the antibodies of the present application (including their variants and derivatives as described herein) and nucleic acids or polynucleotides encoding the antibodies of the present application (including their variants and derivatives as described herein). The antibodies of the present application can also be used to treat, inhibit, or prevent diseases, diseases or conditions, including malignant diseases, diseases, or conditions associated with such diseases or diseases, such as diseases associated with immune responses. In some embodiments, the antibodies of the present application can be used as immunosuppressants. In some embodiments, the antibodies of the present application can be used to treat autoimmune diseases. The antigen-binding polypeptides, variants or derivatives thereof of the present application can be used to inhibit the growth, progression and / or metastasis of cancer, particularly those described in the paragraphs above or below.

[0049] The antibodies or variants or derivatives thereof of the present application can be used to treat, prevent, diagnose and / or predict other diseases or conditions associated with increased cell survival, including, but not limited to, cancer or tumors (including the development and / or metastasis of malignant tumors) and related diseases (e.g., malignant ascites, malignant pleural effusion, nausea and vomiting), such as EpCAM positive tumors.

[0050] Methods of administering the antibody, variant or derivative thereof include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural and oral routes. The antibody or composition can be administered by any convenient route, for example, injection or bolus injection, absorbed through the epithelium or mucosa into the skin lining (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and can be administered with other bioactive agents. Thus, the antibody-containing pharmaceutical composition of the present application can be administered orally, rectally, parenterally, intracerebrally, intravaginally, intraperitoneally, topically (e.g., as a powder, ointment, drop, transdermal patch), orally, or as an oral or nasal spray. The term "parenteral" as used herein refers to administration patterns including intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion. Administration can be systemic or local. It may also be necessary to administer the antigen-binding polypeptides or compositions of the present application locally to the area in need of treatment, which may be accomplished, for example, by localized irrigation during surgery, topical application, for example, in combination with a wound dressing after surgery, by injection, catheter, suppository, or implant, which may be a porous, non-porous, or gel-like material, including membranes or fibers. Preferably, when administering the proteins (including antibodies) of the present application, care should be taken to use materials that do not absorb the proteins. [Brief description of the drawings]

[0051] [Figure 1] FIG. 1 is a schematic diagram of the structure of YBODY antibody. [Diagram 2] Detection of HCT116 and Jurkat cell binding via bispecific antibodies. A: HCT116+no Jurkat antibody negative control flow graph, where Q1 is CFSE stained Jurkat cells, Q2 is co-binding Jurkat and HCT116 cells, Q3 is PKH26 stained HCT116 cells, and Q4 is unstained cells; B: HCT116+Jurkat+M701A 10μg / ml experimental group flow graph, the characteristic meaning of each quadrant is the same as before; C: Concentration gradient curve of HCT116 and Jurkat cell binding via different antibodies. [Diagram 3] Detection of biological activity of bispecific antibodies (reporter gene system). [Figure 4] Bispecific antibody-mediated in vitro killing detection. A: in vitro killing of B16-EpCAM by M701A; B: in vitro killing of B16 by M701A; C: in vitro killing of HCT116 by M701A; D: in vitro killing of OVCAR-3 by M701A; E: in vitro killing of CHO-K1-huEpCAM by different bispecific antibodies, F: in vitro killing of HCT116 by different bispecific antibodies. [Diagram 5] In vivo efficacy of bispecific antibodies in HCT116 human colon cancer model. A: Tumor volume growth pattern of mice; B: Body weight pattern of mice. [Figure 6] In vivo efficacy of bispecific antibodies in OVCAR-3 human ovarian cancer model. A: Tumor volume growth in mice; B: Body weight in mice. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0052] The method and application of the present invention are described below in conjunction with the accompanying drawings, in which the illustrations are for illustrating the present invention, not for limiting the scope of the present invention. Those skilled in the art can make some simple inferences or substitutions without departing from the concept of the present invention, and all of them should be considered as belonging to the protection scope of the present invention.

[0053] Example 1: Construction of expression vector for bispecific antibodies The bispecific antibody structure targeting EpCAM and CD3 includes an anti-EpCAM binding region and an anti-CD3 binding region, the monovalent unit is a pair of anti-EpCAM heavy and light chains, and the single-chain unit is an ScFv-Fc form of anti-CD3, and is defined as a YBODY structure (Figure 1), in which the VL of anti-CD3 is linked to the hinge region and CH2 via a linker. Here, the heavy chain Fc of the monovalent unit and the Fc of the single-chain unit (with human IgG heavy chain Fc as the backbone) are each modified by amino acid mutation so that they are unlikely to form homodimers and are likely to form heterodimers. Using an existing plasmid or synthetic gene fragment as a template, each chain corresponding to the bispecific antibody was amplified by PCR and overlap PCR, and each antibody chain was cloned into the pcDNA3.1 vector (Invitrogen) by enzyme binding or recombination methods. The specific sequence information of each chain of the antibody is shown in Table 1 and the sequence table. [Table 8]

[0054] Example 2: Expression and purification of bispecific antibodies Plasmids were extracted by conventional plasmid extraction methods and used for chemical transfection of CHO-S cells (from Gibco). After transfection, cells were subjected to suspension rocking culture in a rocking platform at 37°C and 5% CO2 for 7-10 days. The supernatant was collected by centrifugation at 3000g and filtered through a 0.22μm filter membrane. Preliminarily purified bispecific antibodies were obtained by protein A affinity chromatography, and the purified protein concentration was measured by UV absorbance at 280nm and the corresponding extinction coefficient, and the antibody purity was tested by high performance size exclusion chromatography (HPLC-SEC), and the corresponding expression amount of each protein was calculated. The expression level of bispecific antibody molecules was 40mg / L-91mg / L, and the initial purity was 45%-81%. The expression levels and initial purity of M701A, M701B, M701C, M701D, M701E, M701F, M701G, M701H, M701I, M701J and M701K are all obviously superior to M701. The affinity samples were then purified by cation exchange chromatography to finally obtain bispecific antibodies with HPLC-SEC purity >95%. The purification recovery rate of each bispecific antibody is shown in Table 2. [Table 9]

[0055] Example 3: Thermal stability detection of bispecific antibodies The purified samples of each bispecific antibody were diluted to 0.5 mg / ml with a buffer (25 mM citric acid + 50 mM NaCl, pH 6.0), dispensed into 1.5 mL EP tubes at 100 μL / tube, and placed in a 40°C water bath for 14 days for a thermal acceleration experiment to detect changes in purity and affinity. The day of placing in the 40°C water bath was counted as D0, and the 14th day was counted as D14.

[0056] Amino coupling method was used to immobilize human EpCAM (SB, Cat: 10694-H08H) and human CD3 antigen (SB, Cat: CT038-H2508H) on CM5 chip, antigen coupling amount was 1500RU, when detecting antigen end binding activity, the sample was diluted to the starting concentration with 1×HBS-EP+buffer, and further diluted to four concentrations with a 2-fold gradient, and detected from low to high concentrations by machine, binding flow rate was 30μL / min, binding time was 120s, dissociation time was 300s, and pH1.5 Glycine solution regeneration chip was used, regeneration flow rate was 10μL / min, regeneration time was 30s. After detection, the software Biacore T200 Evaluation Software was used to perform data fitting on the result spectrum with 1:1 Binding fitting method, and the dissociation equilibrium constant (KD) was obtained.

[0057] The results, as shown in Table 3, showed that the purity loss of M701A, M701B, M701H, M701I, M701J, and M701K on the 14th day of thermal acceleration was less than 5%, of which the purity change of M701A, M701B, M701J, and M701K was less than 2%, and the affinity of both ends remained basically unchanged, indicating that M701A, M701B, M701J, and M701K have good thermal stability. [Table 10]

[0058] Example 4: Bispecific antibody EpCAM end cell affinity detection Using the FACS method, human colon cancer cells HCT116 (Shanghai Institutes of Biological Sciences, Chinese Academy of Sciences) were used as positive cells expressing human EpCAM on the cell membrane surface, and the affinity of the antibody to human EpCAM on the cell surface was detected.

[0059] HCT116 cells were collected by centrifugation, resuspended in buffer (PBS + 1% FBS), and diluted to 2 × 10 550μL per well was added to a 96-well plate at 1000nM cells / well. After centrifugation at 350×g for 5 min, the supernatant was removed. The double antibody was diluted to 1000nM in buffer, diluted 2-fold, added to a 96-well plate at 50μL / well, resuspended, and incubated for 1h away from light, centrifuged, the supernatant was removed, washed twice with buffer, resuspended in diluted PE-labeled anti-human IgG Fc antibody (Biolegend, 409304), incubated for 30min away from light, washed twice with buffer, resuspended in 100μL buffer, and analyzed by flow cytometry (BD Accurio). TM The detection was performed by upgrading to C6).

[0060] Each bispecific antibody had significant binding activity with HCT116 cells, where M700 was the anti-EpCAM terminal mAb control (light chain SEQ ID NO:25 and heavy chain SEQ ID NO:26), and the specific EC50 values ​​were shown in Table 4. [Table 11]

[0061] Example 5: Detection of CD3-terminal affinity of bispecific antibodies (Biacore) Amino coupling method was used to immobilize human CD3 antigen (SB, Cat: CT038-H2508H) on CM5 chip, antigen coupling amount was 1500RU, when CD3 antigen end binding activity was detected, the sample was diluted to the starting concentration with 1×HBS-EP+buffer, and further diluted to four concentrations with a 2-fold gradient, and detected from low to high concentration by machine, binding flow rate was 30μL / min, binding time was 120s, dissociation time was 300s, and pH1.5 Glycine solution regeneration chip was used, regeneration flow rate was 10μL / min, regeneration time was 30s. After detection, the software Biacore T200 Evaluation Software was used to perform data fitting on the result spectrum with 1:1 Binding fitting method to obtain the dissociation equilibrium constant (KD). As shown in Table 5, all of the series bispecific antibodies have binding activity with human CD3 antigen. Here, the anti-EpCAM antibody variable region sequences of M701A and M701B are the same (SEQ ID NO:13 and SEQ ID NO:14), the anti-CD3 antibody variable region sequences are SEQ ID NO:18 and SEQ ID NO:19, respectively, and the corresponding affinities are 21.15nM and 28.27nM, respectively. However, the anti-EpCAM antibody variable region sequences are different (SEQ ID NO:15 and SEQ ID NO:16), the anti-CD3 antibody variable region sequences are also SEQ ID NO:18 and SEQ ID NO:19, and the corresponding affinities of the bispecific antibodies M701H and M701I are 95.26nM and 40.03nM, respectively. This explains the fact that the bispecific antibodies are formed by combining different anti-EpCAM antibodies and different anti-CD3 antibodies, and there is no regularity in the expression of affinity. [Table 12]

[0062] Example 6: Cell-bridging action via bispecific antibodies The EpCAM-positive cell line HCT116 was stained with PKH26, and the CD3-positive cell line Jurkat (Shanghai Institutes of Biological Sciences, Chinese Academy of Sciences) was stained with CFSE. After staining, the cells were cultured at a ratio of 1:1 (1 × 10 5HCT116 cells: 1 × 10 5 Jurkat cells) were added with serially diluted antibodies to be measured, mixed and incubated, where M700 is an anti-EpCAM mAb control (light chain SEQ ID NO:25 and heavy chain SEQ ID NO:26), M100 is an anti-CD3 mAb control (light chain SEQ ID NO:27 and heavy chain SEQ ID NO:28), and Mco101 is an anti-luciferase and anti-CD3 bispecific antibody, which serves as a CD3 terminal isotype control for the bispecific antibody (light chain SEQ ID NO:29, heavy chain SEQ ID NO:30 and single chain SEQ ID NO:31, structure is the same as in Figure 1). After incubation for 1 h, washing and resuspension, the cells were analyzed by flow cytometry (BD Accuri TM C6) and CFSE and PKH26 double positive cells were antibody cross-linked HCT116 and Jurkat cells.

[0063] The results, as shown in Figure 2, showed that after mixing Jurkat and HCT116 cells at a 1:1 ratio and adding hIgG, M700, M100, and Mco101 for 1 hour, HCT116 and Jurkat cells were not crosslinked; after adding M701A and M701B, the crosslinked cells accounted for approximately 40% of the total number of PKH26-positive cells, the activity of M701A and M701B was considerable, and the bispecific antibody-mediated cell interaction and antibody concentration showed a positive quantitative-effect relationship.

[0064] Example 7: Detection of biological activity of bispecific antibodies (reporter gene method) The biological activity of the bispecific antibody was detected using Jurkat-CD3-NFAT-RE-Luc cells (Promega). The pLV-puro (Inovogen Tech.Co., cat.No.VL3001) vector containing DNA encoding the human EpCAM gene (NCBI sequence number: NM_002354.3) was transfected into CHO-K1 cells to obtain a cell line CHO-K1-huEpCAM stably expressing human EpCAM. CHO-K1-huEpCAM was harvested as target cells, resuspended in buffer (PBS + 1% FBS), and 4 × 104 Add 6 × 10 cells / well to an all-white 96-well culture plate, place at 37 °C, and incubate overnight in a 5% CO2 incubator for 18-24 hours, remove the medium in the plate, and add 40 μL of antibody dilution solution per well. Jurkat-CD3-NFAT-RE-Luc cells, i.e., effector cells, were removed, blown off, and a single-cell suspension was made. Add 40 μL, i.e., 6 × 10 cells / well to an all-white 96-well culture plate with an effector-target ratio E:T = 1.5:1. 4 The wells were plated at 100x the cells / well, the white 96-well culture plate was placed at 37°C and incubated in a 5% CO2 incubator for 6 hours, 80μL Bio-Glo luciferase detection solution was added per well, and the plate was incubated at room temperature for 15 minutes away from light, then placed in a multifunction reader to read the luminescence value by chemiluminescence.

[0065] The results, as shown in Figure 3, showed that in the reporter gene evaluation system, the bispecific antibodies M701, M701A, M701B, M701H, M701I, M701J, and M701K all showed biological activity, and the activity of M701A, M701J, and M701K was stronger than that of M701.

[0066] Example 8: Bispecific antibody-mediated in vitro killing detection The isolated PBMCs were used as effector cells, and EpCAM-expressing cells were used as target cells to detect the in vitro killing effect mediated by the bispecific antibody. The pLV-puro (Inovogen Tech.Co., cat.No.VL3001) vector containing DNA encoding the human EpCAM gene (NCBI sequence number: NM_002354.3) was transfected into mouse melanoma B16 cells (Shanghai Institutes of Biological Sciences, Chinese Academy of Sciences) to obtain a cell line B16-EpCAM stably expressing human EpCAM, where B16 was a negative cell that did not express EpCAM. Other target cells expressing EpCAM included human colon cancer cells HCT116 (Shanghai Institutes of Biological Sciences, Chinese Academy of Sciences), human ovarian cancer cells OVCAR-3 (CCTCC, China Center for Typical Culture Collection) and CHO-K1-huEpCAM. The cells were digested with pancreatin into a single-cell suspension, collected by centrifugation at 300 g for 5 min, and stained with 5 μM 5,6-carboxyfluorescein diacetate, succinimidyl ester (CFSE) (37 °C, 15 min), counted with a Vi-cell cell counter after washing twice with complete medium, and added to 96-well plates according to the experimental design, at 2 × 10 per well. 4 Corresponding concentrations of antibodies were added at 50 μL / well, and hPBMCs were counted in a Cellometer cell counter and added to 96-well plates (2×10 cells / well). 5 The cell culture plate was cultured in a cell incubator for 72 h, and the cells were digested into a single-cell suspension. The cells were then added to a propidium iodide (PI) solution with a final concentration of 1 μg / mL, incubated for 10 min, and then analyzed by flow cytometry (BD Accurio). TM C6), and the percentage of CFSE+PI+ bipositive cells among CFSE+ positive cells was analyzed.

[0067] As shown in Figures 4A and 4B, M701A only had a killing effect on B16-EpCAM cells with EpCAM expression, but not on B16 cells without EpCAM expression, and the control antibody Mco101 had no killing effect on any cells, which explained the targeting of the bispecific antibody action. As shown in Figures 4C and 4D, M701A had a clear killing effect on HCT116 and OVCAR-3 cells, which was stronger than the control Mco101. As shown in Figure 4E, the bispecific antibodies M701A, M701B, M701H, and M701I all had a clear killing effect on CHO-K1-huEpCAM cells, and the effects of M701A and M701B were obviously stronger than those of M701H and M701I. As shown in Figure 4F, the bispecific antibodies M701J and M701K had obvious killing effects on HCT116 cells, with the EC50 values ​​ranging from 7.814 to 25.43 ng / ml, and the killing level of M701J was not significantly different from that of M701A. The killing activities of the bispecific antibodies were all significantly stronger than that of M701 (EC50 of M701 was 69.17 ng / ml).

[0068] Example 9: In vivo efficacy of bispecific antibodies in a heterotopic xenograft tumor model of HCT116 human-derived colon cancer According to the culture conditions, a sufficient amount of HCT116 cells and effector cells CIK (cytokine-induced killer, a heterogeneous cell population obtained after co-culturing human peripheral blood mononuclear cells in vitro with multiple cytokines for a certain period of time. This type of cell is also called NK cell-like T lymphocyte because it simultaneously expresses two types of membrane protein molecules, CD3+ and CD56+) were cultured, and the cells were collected and counted. Each mouse was injected with premixed HCT116 cells (2 × 10 6 cells / animal) and CIK (2 × 10 6A human-derived colon cancer HCT116 xenograft tumor model was established by inoculating 10 ...

[0069] Tumor size calculation formula: Tumor volume (mm 3 ) = 0.5 × (tumor long diameter × tumor short diameter 2 ).

[0070] Relative tumor inhibition rate TGI (%): TGI = 1-T / C (%). T and C are the tumor volumes (TV) at a certain time point in the treatment group and control group, respectively. The calculation formula is as follows: T / C% = T TV / C TV ×100%(T TV : Mean tumor volume of treatment group; C TV : Mean tumor volume of the vehicle control group).

[0071] Complete regression rate: Tumor volume is 63mm during or after treatment 3 Tumor complete regression rate (%) = number of animals in a group that achieved complete regression / total number of animals in that group × 100%.

[0072] As shown in Figure 5A, the test drug M701A (2 mg / kg, 1 mg / kg) showed a significant tumor suppression effect in the treatment group on the 30th day after withdrawal (i.e., 33 days after inoculation), with the relative tumor suppression rates TGI (%) being 100% and 93.82%, respectively. The relative vehicle control group was statistically significantly different (p values ​​are both <0.001), and all tumors in the M701A (2 mg / kg) group in both groups reached the complete eradication standard. The efficacy of M701A at both doses was obviously superior to that of M700 at 2 mg / kg (p value <0.001), and the efficacy of M701A at the same dose was obviously superior to that of M701 (both 1 mg / kg). As shown in Figure 5B, there was no weight loss in the animals during the treatment process, and no expression of drug toxicity was observed.

[0073] In the same tumor model, M701B, M701J and M701K showed similar tumor-suppressing effects as M701A at the same dose, and did not reduce body weight.

[0074] Example 10: In vivo efficacy of bispecific antibodies in the OVCAR-3 human-derived ovarian cancer heterotopic xenograft tumor model A sufficient amount of OVCAR-3 cells and effector CIK cells were cultured according to the culture conditions, and the cells were harvested and counted. Premixed OVCAR-3 cells (1 × 10 7 cells / animal) and CIK (1 × 10 7 A human-derived ovarian cancer xenograft tumor model was established by inoculating mice with 1000 cells / mouse (0.1 ml / mouse) at an inoculation volume of 0.2 ml / mouse and 50% Matrigel gel content (0.1 ml / mouse). Treatment was administered 1 h after inoculation. The experiment was divided into a test drug M701A (5 mg / kg), CD3 terminal isotype control Mco101 (5 mg / kg), mab control M700 (5 mg / kg) group and a solvent control group (physiological saline), with 8 mice in each group. The mice were administered intravenously into the tail vein three times on days 0, 2, and 4 after inoculation, respectively. The therapeutic efficacy was evaluated based on the relative tumor inhibition rate (TGI) and complete tumor regression rate, and the safety was evaluated based on the weight change and death status of the animals.

[0075] As shown in Figure 6A, the test drug M701A (5 mg / kg) showed a significant tumor suppression effect in the treatment group on the 44th day after withdrawal (i.e., 48 days after inoculation), with a relative tumor suppression rate TGI (%) of 98.97%. The relative solvent control group had statistically significant differences (all p values ​​<0.001), and the complete tumor elimination rate of the M701A (5 mg / kg) group was 87.5%, and the efficacy of this group was significantly superior to that of 5 mg / kg M700 (TGI = 70.42%) (p value <0.001) and 5 mg / kg Mco101 (TGI = 68.87%) (p value <0.001). As shown in Figure 6B, there was no weight loss of the animals during the treatment process, and no expression of drug toxicity was observed.

[0076] In the same tumor model, M701B, M701J and M701K showed similar tumor-suppressing effects as M701A at the same dose, and did not reduce body weight.

[0077] All documents mentioned in this application are incorporated herein by reference as if each document were incorporated by reference alone. It should also be understood that after reading the above teachings of the present invention, one skilled in the art may make various changes or modifications to the present invention, and equivalents thereof are within the scope defined by the appended claims of this application. [Table 13] TIFF2024540461000017.tif236169 TIFF2024540461000018.tif236169 TIFF2024540461000019.tif245169 TIFF2024540461000020.tif245169 TIFF2024540461000021.tif245169 TIFF2024540461000022.tif245169 TIFF2024540461000023.tif136169

Table 14

Claims

1. a bispecific antibody, comprising an antigen-binding domain that specifically binds to EpCAM and an antigen-binding domain that specifically binds to CD3; wherein the antigen-binding domain that specifically binds to EpCAM comprises a light chain and a heavy chain, or is a light chain-heavy chain pair consisting of them, wherein the light chain comprises a light chain variable region and a light chain constant region, the heavy chain comprises a heavy chain variable region, CH1, and a first Fc fragment, and the first Fc fragment comprises a hinge region, CH2, and CH3a; An antigen-binding domain that specifically binds to EpCAM comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3: According to the Kabat sequence number system, the sequence of CDRL1 is shown in SEQ ID NO: 32, the sequence of CDRL2 is shown in SEQ ID NO: 33, the sequence of CDRL3 is shown in SEQ ID NO: 34, the sequence of CDRH1 is shown in SEQ ID NO: 35, the sequence of CDRH2 is shown in SEQ ID NO: 36, and the sequence of CDRH3 is shown in SEQ ID NO: 37; the antigen-binding domain that specifically binds to CD3 comprises an ScFv that specifically binds to CD3 and a second Fc fragment, or is a fusion peptide consisting of them, wherein the ScFv comprises, in order from the N-terminus to the C-terminus, a heavy chain variable region, a connecting peptide, and a light chain variable region; the second Fc fragment comprises, in order from the N-terminus to the C-terminus, a hinge region, CH2, and CH3b; and the C-terminus of the light chain variable region and the hinge region of the second Fc fragment are linked by the connecting peptide; 1) The antigen-binding domain that specifically binds to CD3 comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3: According to the Kabat sequence numbering system, the sequence of CDRH1 is set forth in SEQ ID NO: 44, the sequence of CDRH2 is set forth in SEQ ID NO: 45, the sequence of CDRH3 is set forth in SEQ ID NO: 46, the sequence of CDRL1 is set forth in SEQ ID NO: 47, the sequence of CDRL2 is set forth in SEQ ID NO: 48, and the sequence of CDRL3 is set forth in SEQ ID NO: 49; or 2) the antigen-binding domain that specifically binds to CD3 comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3; According to the Kabat sequence number system, the sequence of CDRH1 is shown in SEQ ID NO: 52, the sequence of CDRH2 is shown in SEQ ID NO: 53, the sequence of CDRH3 is shown in SEQ ID NO: 54, the sequence of CDRL1 is shown in SEQ ID NO: 55, the sequence of CDRL2 is shown in SEQ ID NO: 56, and the sequence of CDRL3 is shown in SEQ ID NO:

57. Bispecific antibodies.

2. The antigen-binding domain that specifically binds to EpCAM is a heavy chain variable region set forth in SEQ ID NO: 14 or a variant thereof having at least 90% identity to SEQ ID NO: 14, and a light chain variable region set forth in SEQ ID NO: 13 or a variant thereof having at least 90% identity to SEQ ID NO: 13; or The antigen-binding domain that specifically binds to CD3 is (1) a heavy chain variable region set forth in SEQ ID NO: 50 or a variant thereof having at least 90% identity to SEQ ID NO: 50, and a light chain variable region set forth in SEQ ID NO: 51 or a variant thereof having at least 90% identity to SEQ ID NO: 51; or (2) A heavy chain variable region set forth in SEQ ID NO: 58 or a variant thereof having at least 90% identity to SEQ ID NO: 58, and a light chain variable region set forth in SEQ ID NO: 59 or a variant thereof having at least 90% identity to SEQ ID NO: 59; The bispecific antibody of claim 1.

3. The antigen-binding domain that specifically binds to EpCAM comprises: comprising a heavy chain variable region as set forth in SEQ ID NO: 14 or a variant thereof having at least 90% identity to SEQ ID NO: 14; and The antigen-binding domain that specifically binds to CD3 is (1) ScFv as set forth in SEQ ID NO: 18 or a variant thereof having at least 90% identity to SEQ ID NO: 18; and (2) ScFv shown in SEQ ID NO: 19 or a variant thereof having at least 90% identity to SEQ ID NO: 19; The bispecific antibody of claim 1.

4. The bispecific antibody comprises: (1) a light chain constant region shown in any one of SEQ ID NOs: 1, 60-65, and CH1 shown in SEQ ID NO: 2, wherein the first Fc fragment comprises a hinge region shown in SEQ ID NO: 3, a CH2 shown in any one of SEQ ID NOs: 6, 7, 66-71, and CH3a; and (2) A fusion peptide that specifically binds to CD3, comprising or consisting of an ScFv that specifically binds to CD3 and a second Fc fragment, wherein the ScFv comprises a connecting peptide shown in SEQ ID NO: 4, a hinge region shown in SEQ ID NO: 3, a CH2 and a CH3b shown in any one of SEQ ID NOs: 6, 7, 66 to 71, and the C-terminus of the light chain variable region and the hinge region of the second Fc fragment are linked by a connecting peptide shown in SEQ ID NO:

5. The bispecific antibody of claim 1 or 2. Claim 5: The first Fc fragment and the second Fc fragment are human or humanized Fc fragments; or Compared to a wild-type antibody, the first Fc fragment and / or the second Fc fragment comprises one or more substitutions that form a knob-into-hole pair between the heavy chain and the fusion peptide, i.e., T366 in one CH3 domain is substituted with tyrosine (Y) or tryptophan (W), and Y407 in the other CH3 domain is substituted with threonine (T), alanine (A), or valine (V); or The first Fc fragment and / or the second Fc fragment comprises one or more substitutions, 1) the substitutions form salt bridge pairs between the heavy chain and the fusion peptide, i.e., one CH3 domain comprises substitutions with one or more amino acid residues that are positively charged under physiological conditions, and the other CH3 domain comprises substitutions with one or more amino acid residues that are negatively charged under physiological conditions, the positively charged amino acid residues being arginine (R), histidine (H), or lysine (K), and the negatively charged amino acid residues are substituted with one or more amino acid residues that are negatively charged under physiological conditions, the positively charged amino acid residues being arginine (R), histidine (H), or lysine (K). the base is aspartic acid (D) or glutamic acid (E), and the substituted amino acid residues include one or more of D356, L368, K392, D399, and K409 in Table 7, 2) the substitution forms a disulfide bond between the heavy chain and the fusion peptide and is a substitution in Table 8, and / or 3) the substitution significantly reduces the binding ability between Fc and Protein A, i.e., H435 and Y436 on one CH3 domain are substituted with arginine and phenylalanine, respectively, as shown in Table 9; or The sequences of CH3a and CH3b are selected from the group consisting of: (1) one sequence of which is shown in SEQ ID NO: 8 and another sequence of which is shown in SEQ ID NO: 11; (2) one of the sequences is shown in SEQ ID NO: 9 and another is shown in SEQ ID NO: 12; (3) one sequence of which is shown in SEQ ID NO: 72 and another sequence of which is shown in SEQ ID NO: 74; (4) one sequence of which is shown in SEQ ID NO: 9 and another sequence of which is shown in SEQ ID NO: 75; and (5) One sequence of which is shown in SEQ ID NO: 73 and another sequence of which is shown in SEQ ID NO: 76; The bispecific antibody of claim 3.

6. The bispecific antibody is selected from the group consisting of: (1) A bispecific antibody comprising or consisting of a fusion peptide, a heavy chain, and a light chain, wherein the fusion peptide comprises or consists of SEQ ID NO: 18, SEQ ID NO: 5, SEQ ID NO: 3, SEQ ID NO: 6, and SEQ ID NO: 11, the heavy chain comprises or consists of SEQ ID NO: 14, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 6, and SEQ ID NO: 8, and the light chain comprises or consists of SEQ ID NO: 13 and SEQ ID NO: 1; (2) Bispecific antibodies comprising or consisting of a fusion peptide, a heavy chain, and a light chain, wherein the fusion peptide comprises or consists of SEQ ID NO: 19, SEQ ID NO: 5, SEQ ID NO: 3, SEQ ID NO: 6, and SEQ ID NO: 11, the heavy chain comprises or consists of SEQ ID NO: 14, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 6, and SEQ ID NO: 8, and the light chain comprises or consists of SEQ ID NO: 13 and SEQ ID NO: 1; (3) Bispecific antibodies comprising or consisting of a fusion peptide, a heavy chain, and a light chain, wherein the fusion peptide comprises or consists of SEQ ID NO: 18, SEQ ID NO: 5, SEQ ID NO: 3, SEQ ID NO: 7, and SEQ ID NO: 12, the heavy chain comprises or consists of SEQ ID NO: 14, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 7, and SEQ ID NO: 9, and the light chain comprises or consists of SEQ ID NO: 13 and SEQ ID NO: 1; (4) A bispecific antibody comprising or consisting of a fusion peptide, a heavy chain, and a light chain, wherein the fusion peptide comprises or consists of SEQ ID NO: 19, SEQ ID NO: 5, SEQ ID NO: 3, SEQ ID NO: 7, and SEQ ID NO: 12, the heavy chain comprises or consists of SEQ ID NO: 14, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 7, and SEQ ID NO: 9, and the light chain comprises or consists of SEQ ID NO: 13 and SEQ ID NO: 1; (5) A bispecific antibody comprising or consisting of a fusion peptide, a heavy chain, and a light chain, wherein the fusion peptide comprises or consists of SEQ ID NO: 18, SEQ ID NO: 5, SEQ ID NO: 3, SEQ ID NO: 6, and SEQ ID NO: 8, the heavy chain comprises or consists of SEQ ID NO: 14, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 6, and SEQ ID NO: 11, and the light chain comprises or consists of SEQ ID NO: 13 and SEQ ID NO: 1; (6) A bispecific antibody comprising or consisting of a fusion peptide, a heavy chain, and a light chain, wherein the fusion peptide comprises or consists of SEQ ID NO: 19, SEQ ID NO: 5, SEQ ID NO: 3, SEQ ID NO: 6, and SEQ ID NO: 8, the heavy chain comprises or consists of SEQ ID NO: 14, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 6, and SEQ ID NO: 11, and the light chain comprises or consists of SEQ ID NO: 13 and SEQ ID NO: 1; (7) A bispecific antibody comprising or consisting of a fusion peptide, a heavy chain, and a light chain, wherein the fusion peptide comprises or consists of SEQ ID NO: 18, SEQ ID NO: 5, SEQ ID NO: 3, SEQ ID NO: 7, and SEQ ID NO: 9, the heavy chain comprises or consists of SEQ ID NO: 14, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 7, and SEQ ID NO: 12, and the light chain comprises or consists of SEQ ID NO: 13 and SEQ ID NO: 1; and (8) A bispecific antibody comprising or consisting of a fusion peptide, a heavy chain, and a light chain, wherein the fusion peptide comprises or consists of SEQ ID NO: 19, SEQ ID NO: 5, SEQ ID NO: 3, SEQ ID NO: 7, and SEQ ID NO: 9; the heavy chain comprises or consists of SEQ ID NO: 14, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 7, and SEQ ID NO: 12; and the light chain comprises or consists of SEQ ID NO: 13 and SEQ ID NO: 1; The bispecific antibody of claim 1.

7. A nucleic acid composition comprising a nucleic acid sequence encoding the bispecific antibody of any one of claims 1 to 6.

8. A pharmaceutical composition for treating cancer, malignant ascites, malignant effusion, or malignant pleural effusion, comprising the bispecific antibody of any one of claims 1 to 6 and a pharmaceutically acceptable carrier.

9. The pharmaceutical composition further comprises a drug, or the cancer is an EpCAM-positive tumor, or The pharmaceutical composition according to claim 8, wherein the dosage form of the pharmaceutical composition is a gastrointestinal dosage form or a parenteral dosage form.

10. The cancer is colon cancer, stomach cancer, breast cancer, ovarian cancer, lung cancer, prostate cancer, pancreatic cancer, liver cancer, retinoblastoma, esophageal cancer, renal cancer, renal clear cell tumor, skin squamous cell carcinoma, skin basal cell carcinoma, sarcoma, nasal glioma, craniopharyngeal duct tumor, thyroid cancer, cholangiocarcinoma, bladder cancer, head and neck tumor, cervical cancer, or oral cancer, or 10. The pharmaceutical composition according to claim 9, wherein the dosage form of the pharmaceutical composition is an injection, including intravenous injection, intravenous drip, subcutaneous injection, local injection, intramuscular injection, intratumoral injection, intraperitoneal injection, intracranial injection, or intracavity injection.

11. 7. A conjugate or fusion protein comprising the bispecific antibody of any one of claims 1 to 6 and a substance A conjugated or fused to the bispecific antibody, wherein substance A is selected from the group consisting of a therapeutic agent, a drug precursor, a protein, a virus, a lipid, a biological response modifier, PEG, a hormonal agent, an oligonucleotide, a diagnostic agent, a cytotoxic agent which may be a drug or a toxin, an ultrasound-enhancing agent, a non-radioactive marker, a detectable marker, or a fluorescence-emitting metal.

12. A kit comprising the bispecific antibody of any one of claims 1 to 6.

13. A kit as described in claim 12, further comprising a drug for treating cancer, malignant ascites, malignant effusion, or malignant pleural effusion, wherein the cancer is a cancer described in claim 10.

14. A bispecific antibody according to any one of claims 1 to 6 for the treatment of cancer, malignant ascites, malignant effusion, or malignant pleural effusion, wherein the cancer is a cancer according to claim 10.

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